Squash Trypsin Inhibitors fromMomordica cochinchinensisExhibit an Atypical Macrocyclic Structure

Abstract

Three trypsin inhibitors (TIs), from the seeds of the squash Momordica cochinchinensis (MCo), have been isolated and purified using gel filtration, ion exchange chromatography, and reverse-phase HPLC. Their sequences could be determined only after proteolytic cleavages. In the case of MCoTI-I and -II, it was shown that their polypeptide backbones are cyclic, a structure that has never been described in squash TIs. They contain 34 amino acid residues with 3 disulfide bridges and measured molecular masses of 3453.0 and 3480.7, respectively. They are the largest known macrocyclic peptides containing disulfide bridges. Their sequences show strong homology to other squash TIs, suggesting a similar three-dimensional structure and an analogous mechanism of action. A model of MCoTI-II was constructed by analogy to the crystal structure of the complex between bovine trypsin and CMTI-I, indicating that the linker connecting the two termini is flexible and does not impose significant geometrical constraints. This flexibility allows an Asp-Gly peptide bond rearrangement to occur in this region, giving rise to two isoforms of MCoTI-II. Although the importance of cyclization is not clear, it might confer increased stability and resistance to proteolysis. A minor species, MCoTI-III, was also characterized as containing 30 amino acid residues with a molecular mass of 3379.6. This component possesses a linear backbone with a blocked N-terminus. MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds bearing new binding activities.

Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides

“…MCoTI-II synthesised by this chemoenzymatic method was identical in all respects to naturally occurring MCoTI-II. 18,19,29,36 Interestingly, concomitant release of a small amount (8%) of rf-MCoTI-II is observed, although it was absent from the This facile new route to MCoTI-II was explored in a series of experiments that show that PST possesses ligase specificity for positively charged (Arg/Lys) residues at P 1 in the inhibitory loop. The open-chain forms of the naturally 50 occurring cyclotide MCoTI-I (P 1 =Lys) and three MCoTI-II analogues in which P 1 was changed to Arg, Gln or Phe (Table 2) were synthesised by SPPS, and subjected to the refolding and PST-mediated ligation conditions described above.…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…[5] Cyclotides display a diverse range of biological activities, including uterotonic, [6] haemolytic, [7][8][9] neurotensin antagonistic, [10] HIV inhibitory, [11] antimicrobial, [12] cytotoxic, [13] antifouling, [14] and trypsin inhibitory activities. [15] In addition, kalata B1 and B2 have been shown to inhibit the growth and development of Helicoverpa punctigera and H. armigera larvae, suggesting that cyclotides function as insecticidal agents as part of plant defence systems. [16][17][18][19] A presumed role in plant defence is also supported by recent reports of their molluscidal activity against the Golden Apple snail, a major pest of rice in South East Asia, [20] and also by their anthelmintic activities.…”

Peptídeos cíclicos de biomassa vegetal: características, diversidade, biossíntese e atividades biológicas

“…(Cucurbitaceae). 35,36 Esses peptídeos estão agrupados aos ciclotídeos, mas algumas evidências estruturais tornam essa abordagem contraditória, gerando discussões sobre esta classificação. Peptídeos inibidores de tripsina e sua correlação com os ciclotídeos serão discutidos adiante em maiores detalhes.…”

“…As defensinas são pequenas proteínas (45-54 resíduos de aminoáci-dos) que retêm em sua estrutura oito resíduos de cisteína altamente conservados e que formam quatro ligações dissulfeto, responsáveis pela estabilização de sua estrutura tridimensional. 103 O conjunto dos resíduos de cisteína, bem como a organização das ligações dissulfeto formada é conhecida como "nó de cistina" (Cystine Knot-CK), presente não somente nas defensinas, mas também em fatores de crescimento, inibidores de tripsina (MCoTIs) 35,36 e inibidores de carboxipeptidase A de batata. 104 Distribuídas desde moluscos, insetos, vertebrados e vegetais, as defensinas são moléculas envolvidas na resposta imune inata, um sistema de defesa primitivo no combate às infecções microbianas.…”

“…No entanto, 48-70% de identidade na sequência dos resíduos foram encontrados para outros inibidores de tripsina de Cucurbitaceae. 25,35,36 Assim, a relação evolutiva entre essas suas classes distintas de peptídeos macrocíclicos ainda é obscura merecendo estudos adicionais de biossíntese.…”